S1

BODIPY-based colorimetric/ratiometric fluorescence probes for sulfite in

aqueous solution and in living cells

Shanshan Liu,aLun Song,a Qian Sun,aZhaoyang Chen,a Yu Ge,bWeibing Zhanga and Junhong

Qian*a aShanghai Key Laboratory of Functional Materials Chemistry, School of Chemistry and Molecular Engineering, East China University of Science and Technology, Shanghai 200237, China

bShanghai Institute of Quality Inspection and Technical Shanghai, 200233, China.

junhongqian@ecust.edu.cn

 

Supporting Information

Contents

Experiment--------------------------------------------------------------------------------------S2

NMR spectra of M1 and M2------------------------------------------------------------------S4

NMR and ESI spectra of M3, BSP1 and BSP2--------------------------------------------S5

The absorption and emission spectra of BSP1 in different solvents -------------------S11

The concentration-dependent absorption spectra of BSP1 in PBS --------------------S12

Plot of fluorescence intensity vs. absorbance of BSP1----------------------------------S13

I554/I618 vs. time of BSP1 in the presence of sulfite-------------------------------------S14

The influence of pH value on I554/ I618 of BSP1------------------------------------------S15

The concentration-dependent absorption spectra of BSP2 in PBS --------------------S16

The normalized absorption spectra of BSP1 and BSP2---------------------------------S17

Time-dependent absorption and emission spectra of BSP2 in CTAB-PBS-----------S18

Sulfite effect on the absorption spectrum of BSP2--------------------------------------S19

Electronic Supplementary Material (ESI) for RSC Advances.This journal is © The Royal Society of Chemistry 2015

S2

Experiment

Materials and reagents

All the commercial reagents and solvents (Aladdin Corporation) were of analytical

grade and used without further purification. Ultra-pure water was prepared through a

Sartorius Arium 611DI system. Phosphate salts were used to keep a stable pH and ion

strength in detection systems. Absorption spectra were measured with an Evolution

220 UV-Visible spectrophotometer (Thermo Scientific). Fluorescence spectra were

conducted on a Lumina Fluorescence Spectrometer (Thermo Scientific). All pH

measurements were performed with a model FE20 meter purchased from Mettler

Toledo. NMR spectra were recorded using a Bruker AV-400 spectrometer (400MHz).

Mass spectra were performed with a MA 1212 Instrument using standard condition

(ESI, 70 eV).

Time titration of sulfite-probe systems

3 mM of the stock solutions of BSP1 and BSP2 in DMF were prepared ahead. The

stock solution of BSP1 or BSP2 was diluted with PBS (20 mM, pH 7.4) with or

without 1 mM CTAB to acquire 1×10-5 M dye aqueous solution. 50 μL of 30 mM

freshly prepared Na2SO3 in PBS (20 mM, pH 7.4) were added to 3 mL of 1×10-5 M

dye aqueous solution. Absorption and emission spectra of the above solution were

collected at different intervals.

Sulfite titration

0~50 μL of 30 mM Na2SO3 in PBS were added into 3 mL of 1×10-5 M BSP1 in

CTAB-PBS or BSP2 in PBS solutions. The spectra were recorded 2 h and 20 min

after each addition of sulfite to BSP1 and BSP2 solutions, respectively.

HPLC traces

High-performance liquid chromatography (HPLC) spectra were carried out on an

Agilent Technologies 1260 Infinity LC system. The mobile phases were degassed

with an ultrasonic apparatus for 14 min. Mobile phase: A: water, B: acetonitrile;

gradient elution: 2–8 min 10–95% B, 10-12 min 95–10% B; Isocratic elution: 0–2 min

10% B, 8-10 min 95% B, and 12-14 min 10% B. Injection volume: 10 μL; flow rate:

1.0 mL/min; detection wavelength: isosbestic point 512 nm.

S3

Live cell culture and fluorescence imaging

HeLa cells were cultured in Dulbecco's modified Eagle's medium (DMEM)

supplemented with 10% fetal bovine serum (FBS) in an atmosphere of 5% CO2 and

95% air at 37 °C. The cells were washed with phosphate buffered solution (PBS, 20

mM, pH 7.4) and pre-incubated with 500 μM Na2SO3, then incubated with BSP1 (10

μM) and CTAB (1 mM) in DMEM for 120 min at 37 C and washed 3 times with

PBS. For the control experiment, the cells were only incubated with 10 μM of BSP1

and CTAB (1 mM) for 120 min. Cell imaging was carried out after washing the cells

with PBS. Emission was collected at 500-550 nm for the green channel and at

570–620 nm for the red channel. The excitation wavelength was set at 514 nm for

green and red channels.

S4

Fig. S1 1H-NMR spectra of M1 and M2.

S5

S6

4000 3500 3000 2000 1500 1000 500

20

40

60

80

100

T %

/ cm-1

M3

Fig. S2 1H-NMR, 13C-NMR, MS and IR spectra of M3.

S7

S8

4000 3500 3000 2000 1500 1000 50020

40

60

80

100

/ cm-1

T %

BSP1

Fig. S3 1H-NMR,13C-NMR, MS and IR spectra of BSP1.

S9

S10

4000 3500 3000 1500 1000 50030

40

50

60

70

80

90

100

/ cm-1

T %

BSP2

Fig. S4 1H-NMR, 13C-NMR, MS and IR spectra of BSP2.

S11

400 450 500 550 600 650 700 750

0.00

0.06

0.12

0.18

0.24

0.30

DCM MeCN EA MeOH DMF CTAB-PBS PBS H2OA

/ nm

a

560 600 640 680 7200

8000

16000

24000

32000

40000

I / a

.u.

/ nm

EA DCM DMF MeCN CTAB-PBS MeOH PBS H

2O

b

Fig. S5 The absorption (a) and emission (b) spectra of BSP1 (10 µM) in different

solvents.

S12

400 450 500 550 600 650 700 750

0.000

0.007

0.014

0.021

0.028

0.035

[BSP1] / M 0.1 0.3 0.5 0.7 1 1.5 2 2.5 3 3.5

A

[BSP1] / M

a

0.0 0.8 1.6 2.4 3.2 4.00.000

0.007

0.014

0.021

0.028

0.035

A

[BSP1] / M

b

Fig. S6 The absorption spectra of BSP1 with different concentrations in PBS.

S13

0.05 0.10 0.15 0.20 0.25

3000

6000

9000

12000

15000

I 618 /

a.u.

A554

Fig. S7 Plot of fluorescence intensity vs. absorbance of BSP1.

S14

0 30 60 90 120 150 180

0

1

2

3

4I 55

4 / I 61

8

t / min

Fig. S8 I554/I618 vs. time of BSP1-sulfite system. [BSP1] =10 μM, [sulfite] = 500 M,

1 mM CTAB-PBS (20 mM, pH 7.4), λex= 512 nm, 25°C.

S15

2 4 6 8 10 12

0

2

4

6

13500

15000

16500

0

2

4

6

13500

15000

16500

I 554 /

I 618

I / a

.u.

pH

a

2 4 6 8 10 120.22

0.24

4

8

0.22

0.24

4

8

A49

4 / A

554

A

pH

b

Fig. S9 The effect of pH value on the fluorescence intensity of BSP1 at 618 nm and I554/I618 of BSP1-SO3H (a), and the absorbance of BSP1 at 554 nm and A494/ A554 of BSP1-SO3H (b), 1 mM CTAB-PBS (20 mM).

S16

400 500 600 700

0.00

0.08

0.16

0.24

0.32

0.40

[BSP2] / M 1 3 5 7 10 15 20 25 30

A

/ nm

a

0 6 12 18 24 30

0.00

0.08

0.16

0.24

0.32

0.40

A56

7

[BSP2] / M

b

Fig. S10 The absorption spectra of BSP2 with different concentrations (1~30M)

in PBS.

S17

500 550 600 650 7000.0

0.2

0.4

0.6

0.8

1.0

BSP2 BSP1

A /

norm

aliz

ed

nm

13 nm

Fig. S11 The normalized absorption spectra of BSP1 and BSP2.

S18

400 450 500 550 600 650 700 750

0.00

0.03

0.06

0.09

0.12

t / min 0 1 3 5A

/ nm

a

560 600 640 680 720

0

800

1600

2400

3200

4000

/ nm

t / min 0 1 3 5

I / a

.u.

b

Fig. S12 Time-dependent absorption (a) and emission (b) spectra of BSP2 (10M) in the presence of 500 M of sulfite in 1 mM CTAB-PBS (20 mM, pH 7.4) system, λex= 520 nm.

S19

400 450 500 550 600 650 700

0.00

0.03

0.06

0.09

0.12

[sulfite] / M 0 10 20 40 50 80 100

A

/ nm

a

0 20 40 60 80 100

0.4

0.8

1.2

1.6

2.0

A47

0 / A

567

[sulfite] / M

y=0.2739+0.01468x

R2=0.9844

b

Fig. S13 (a) The absorption spectra of BSP2 with different concentrations of sulfite; (b) the absorbance ratio at 470 nm and 567 nm (A470/ A567) as a function of sulfite concentration. 20 mM PBS, pH 7.4, 25°C, [BSP2] = 10 μM, recorded 20 min after each addition.